This invention relates to a method of plant tissue culture and regeneration. More particularly the invention relates to a method for the genetic transformation of plant cells, the regeneration of whole plants from such cells and the plants so produced. Particularly, but not exclusively, the invention relates to a method for the transformation of Beta vulgaris, which includes sugar beet, fodder beet, table beet and Swiss chard.
Although genetic transformation and subsequent regeneration is largely a matter of routine nowadays for many plants species, some species have remained recalcitrant to transformation by most of the numerous methods which are available. Beta vulgaris is one such example where, despite transient expression in some cells and occasional success with specific genotypes, no simple routine method is available for the production of transgenic plants (International Patent Application No. WO 91/13159; D'Halluin, K. et.al., Biotechnology 10 309-314 (1992)). More particularly, no method for transformation via direct gene transfer and subsequent regeneration has yet been published. The recalcitrance of sugar beet protoplasts is well documented (Lindsey et.al. Transformation in Sugar Beet (Beta vulgaris L.) Biotechnology in Agriculture and Forestry, Vol 23, Plant protoplasts and Genetic Engineering IV" Y. P. S. Bajaj, Ed., Springer-Velag, Berlin, 1993). Cell division in vitro is restricted and totipotent colonies are generally obtained only at low frequency (0.1% or less). Protoplasts isolated from sugar beet leaves vary in size and morphology, reflecting the high degree of cellular heterogeneity present within the source tissue both at physiological (resulting from the relative location of the cells in vivo) and cytogenetic (ploidy, cell cycle phase) levels.
There is, therefore, a continuing need for a simple, high frequency transformation method which may be applicable to beet.
In order that a cell may be efficiently transformed, certain requirements must be satisfied. First the gene to be inserted must be assembled within a construct which contains effective regulatory elements which will drive transcription of the gene. Next, there must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. The probability of integration may be improved by certain means but, in general, integration is simply a matter of uncontrolled chance. Finally, as far as plants are concerned the target cell type must be such that cells can be regenerated into whole plants.
Plant cells are more difficult to transform than bacterial or animal cells because the presence of hard cell walls presents a barrier to insertion of the construct through that wall.
Thus the choice of method for the transformation of plant cells tends to be limited to those which are convenient for the target plant type (Potrykus, I, in Plant Breeding: Principles and Prospects, Ed. Hayward et.al., Publ. Chapman & Hall, London (1993)). As a generalisation, dicotyledonous plants are relatively easy to transform whereas monocotyledonous plants are very difficult, there being only a few techniques available in respect of which success has been reported, and that with very low success rate.
One method which is claimed to transform plant cells is the procedure known as "microinjection" where, under the microscope, a DNA construct is injected from a hollow needle into a target cell. A variant of that procedure is the rupturing of the cell wall with a needle, the DNA being added to the surrounding medium and allowed to diffuse into the cell through the break in the cell wall. This variant is known as "micropricking". Both of these procedures require a high degree of manipulative skill by the operator and are very time consuming. Japanese Published Patent Application Number 03103183 of 1991 proposes that a foreign gene may be inserted into plant cells by microinjection into guard cells which occur in the epidermal tissue of a plant, followed by culture of those cells. However, physiological studies of guard cells have shown that they have abnormally high intracellular pressures and it is reasonable to question whether microinjection of DNA into guard cells is feasible and, in any case this method would suffer from the principal drawback of microinjection which is that the process is so time-consuming that only a relatively small number of cells can be injected in the course of a day. No data were presented which accurately describe the isolation of transgenic plants by this route.
It is probably true to say that to date the most effective methodology for the introduction of gene constructs, particularly for monocotyledonous cells has been the so-called "biolistics" method in which high density metallic particles, usually of tungsten or gold, are coated with the gene construct and are propelled by an explosive release of gas at a target cell culture. This alternative approach abandons the high precision of targeting which is inherent in microinjection and micropricking, in favour of a rapid "pepperpot" approach which enables large numbers of cells to be "hit" in a short time, giving a large number of putative transformants for screening.
Effective though the biolistic method may be, it requires expensive hardware and, although rapid by comparison with some other methods which have been attempted, is time-consuming. It does, however, achieve high numbers of transformation events at each bombardment. One problem with this technique is the effect of the blast of expanding gas on the target tissue. Another is the difficulty of aiming the projectile shower at a selected area of the target. Various microtargeting devices have been designed to help overcome this latter problem (Leduc et.al., Sex.Plant Reprod., 7, 135-143 (1994); Iglesias et.al., Planta, 192, 84-91 (1994)) but to date no transgenic plants have been produced.
Mixing of plant cells with plasmid DNA and sub-micron diameter fibres or whiskers is a simple and inexpensive alternative transformation method. There have been several published reports of transformation using silicon carbide whiskers. The first described transient expression of .beta.-glucuronidase (gus) in Black Mexican Sweet (BMS) corn suspension cells (Kaeppler et al., 1990). The same group have recently published their results on stable transformation of BMS and tobacco (Kaeppler et al., 1992). In the corn system a mean of 3.4 BASTA-resistant BMS colonies were recovered from each vortex-treated sample of cells (300 .mu.l packed cell volume) using a BAR and gus-containing plasmid. Sixty five per cent of these herbicide-resistant colonies expressed gus. (Kaeppler H. F., Gu W., Somers D. A., Rines H. W, Cockburn A. F. (1990) "Silicon carbide fiber-mediated DNA delivery into plant cells", Plant Cell Reports 9: 415-418, and, Kaeppler H. F., Somers D. A., Rines H. W., Cockburn A. F. (1992), "Silicon carbide fiber-mediated stable transformation of plant cells", Theor. Appl. Genet. 84: 560-566. The use of whiskers for the transformation of plant cells, particularly maize, is the subject of U.S. Pat. No. 5,302,523 in the name of Zeneca Limited.
There are numerous factors which influence the success of transformation. The design and construction of the exogenous gene construct and its regulatory elements influence the integration of the exogenous sequence into the chromosomal DNA of the plant nucleus and the ability of the transgene to be expressed by the cell. A suitable method for introducing the exogenous gene construct into the plant cell nucleus in a non-lethal manner is essential. Importantly, the type of cell into which the construct is introduced must, if whole plants are to be recovered, be of a type which is amenable to regeneration, given an appropriate regeneration protocol.